Responsive paramagnetic mri contrast agents

ABSTRACT

A method is claimed based on CEST procedure for the in vivo or in vitro determination of physical or chemical parameters which includes the use of a responsive paramagnetic CEST contrast agent.

This invention refers to a method based on CEST procedure for the invivo, ex vivo, or in vitro determination of physical or chemicalparameters of diagnostic interest comprising the use of at least oneCEST responsive paramagnetic contrast agent.

BACKGROUND OF THE INVENTION

It is now well established that the potential of Magnetic ResonanceImaging (MRI) procedures can be further enhanced when this diagnosticmodality is applied in conjunction with the administration of contrastagents (CAs), i.e. chemicals able to promote marked changes in therelaxation rates of the tissue protons. According to the major effectsthey produce on images, CAs are classified as positive or negativeagents. The positive CAs are represented by paramagnetic complexes,mostly containing Gd(III) or Mn(II) ions, which affect the relaxationrates of the bulk water through the exchange of the water molecules intheir coordination spheres (Caravan P, et al. Chem Rev 1999,99:2293-2352; the Chemistry of Contrast Agents in Medical MagneticResonance Imaging. Chichester, UK: John Wiley & Sons; 2001. p 45-120).Their effect is similar on T₁ and T₂ but, being T₁ usually significantlylonger than T₂ in most biological tissues, their effect is more oftenexploited in T₁-weighted images, thus resulting in brighter spots in theimages.

On the contrary, negative CAs are used to shorten T₂, leading to animproved contrast by reducing the water signal in T₂-weighted images.

Furthermore, it was early reported that chemicals containing mobileprotons may act as T₂-agents through the reduction of the water protonrelaxation time via exchange processes (Aime S et al., Invest Radiol1988; 23(Suppl 1):S267-S2706).

A different way to efficiently reduce water signal occurs when a properradiofrequency pulse (rf) is applied at the resonance frequency of anexchangeable proton saturating it. This results in a net decrease of thebulk water signal intensity owing to a saturation transfer effect.

This alternative MRI contrast-enhancing technique is named ChemicalExchange Dependent Saturation Transfer (CEDST or, more commonly, CEST)(Balaban RS.: Young IR, editor. Methods in Biomedical Magnetic ResonanceImaging and Spectroscopy. Chichester, UK: John Wiley & Sons; 2000.Vol. 1. p 661-6667).

Suitable contrast agents for this technique include at least onechemically exchangeable proton.

The efficacy of most prior art contrast agents, either the conventionalT₁ and T₂-reducing MRI contrast agents or the CEST agents, is related tothe different cellular uptake of the administered complex compound or tothe different distribution thereof through the extracellular spaces ofthe targeted organ or tissue. No contrast is detectable if the uptakebetween the target and the surrounding tissue is similar.

Moreover, the prior art contrast agents are expressly addressed to theproduction of images of the targeted tissue or organ and, generally, areunable to act as reporter of specific physical or chemical parameters ofthe examined tissue which could represent a quantitative assessment of aphysio/pathological state.

Otherwise, WO 00/66180 discloses a method for enhancing the contrast ofMRI images which comprise the administration of a CEST MRI contrastagent including at least one chemical group endowed with appropriateproton exchange and chemical shift properties to function effectivelyfor performing CEST MRI analyses in vivo as well as for the thedetermination of physical or chemical parameters such as pH andtemperature both in vivo and in vitro. A ratiometric method for the pHmeasurement which is independent on the contrast agent concentration isalso disclosed.

All the agents disclosed by Balaban and co-workers as useful to practicethe claimed method are diamagnetic organic molecules having OH or NHexchangeable protons.

In general, the mobile protons of a contrast agent for a CESTapplication must possess a fast exchange rate (k_(ex)) with waterprotons, but slower than the coalescence condition, wherein thiscondition is suitably reached when k_(ex)Δv˜1/2π, where Δv is thechemical shift separation in Hz between the two exchanging pools. Inthis context, larger Δv values enable the exploitation of higher k_(ex)values, thus resulting in an enhanced CEST effect.

The diamagnetic systems claimed by Balaban are advantageously endowedwith adequately short relaxation rates, but the chemical shiftsseparation from their NH or OH exchangeable protons signals and the bulkwater signal is only within 1-5 ppm. So, the saturation of these mobileprotons, avoiding the saturation of the bulk water or protein boundwater, could actually present a considerable difficulty. Beside thesmall Δv values, a further limit of such diamagnetic agents isrepresented by the high concentration thereof which is usually requiredto generate a sufficiently large CEST effect, resulting in a highprobability of toxic or physiological effect in vivo.

WO 02/43775 discloses paramagnetic metal ion-based macrocyclic CESTcontrast agents which comprises a tetraazacyclododecane ligand whereinpendent arms includes amide groups, a paramagnetic metal ion coordinatedto the ligand and a water molecule associated with it. Said agents arereported to be useful for producing image contrast based on amagnetization transfer mechanism.

The specification cites the effect of the pH on the residence lifetimeat 298 K, τ_(M) ²⁹⁸, (τ_(M)=1/k_(ex)) for protons associated with theamides in the pendent arms and the pH dependence of the MagnetizationTransfer effect obtained while saturating two magnetically differentexchangeable protons associated to the same amide group of one ofclaimed compounds (FIG. 25 and experiment 13, respectively). Thespecification, however, fails to teach or even suggest the applicabilityof the pH effect on the magnetization transfer to the whole class ofclaimed agents. Moreover, either the specification or the experiment 13fail to teach or to suggest the possible use of the claimed compounds ina method of general applicability for the determination of a physical orchemical parameter of diagnostic interest in a human or animal bodyorgan, fluid or tissue; even less in a method wherein said determinationis obtained independently on the local contrast agent concentration. Atthe same time, WO 02/43775 specification fails to teach how saiddetermination may be carried out.

SUMMARY OF THE INVENTION

The present invention relates to a method based on the CEST procedurefor the in vivo, in vitro or ex vivo determination of a physical orchemical parameter of diagnostic interest which includes theadministration of a responsive paramagnetic CEST contrast agent.

In particular, it is an object of the present invention a method for thedetermination, by use of the Magnetic Resonance Imaging technique, of aphysical or chemical parameter of diagnostic interest in a human oranimal body organ, fluid or tissue wherein:

-   -   a responsive paramagnetic CEST contrast agent is employed        comprising at least one exchangeable proton whose saturation        capability is correlated to the physical or chemical parameter        of interest, and    -   a CEST MR image which is responsive for said parameter in the        organ or tissue under examination is registered.

The paramagnetic contrast agent for use in the method of the inventionis a responsive agent, i.e. an agent which combines the characterisingfeatures of a CEST agent with the fact that the saturation transfereffect that it enables is sensitive to the physical or chemicalparameter of diagnostic interest. Accordingly, the contrast agent foruse in the method of the invention is a paramagnetic compound whichcomprises at least one mobile proton in chemical exchange with the watermedium protons and which is able, when a proper radiofrequencyirradiating field is applied at the resonance frequency of the saidexchangeable proton, to generate a saturation transfer (ST) effectbetween said mobile proton and the water medium protons that onlycorrelates to the physico-chemical parameter of diagnostic interest.

In a preferred method according to the invention, a CEST paramagneticcontrast agent is administered which is endowed with at least twomagnetically different mobile protons or proton pools allowing theregistration of CEST MR image responsive for the physico-chemicalparameter of diagnostic interest and independent on the administeredcontrast agent concentration.

The responsive paramagnetic CEST contrast agents for use in the methodof the invention overcome the limits affecting the prior-art diamagneticcompounds of WO 00/66180. The molecular structure of the paramagneticresponsive agents according to the present invention, in fact, canadvantageously be selected in order to pursue optimal values for thechemical shifts and exchange rates of the mobile protons with waterprotons. Further, the structures of the diamagnetic agents of WO00/66180 include a relatively small number of mobile protons which cannot be easily increased. Conversely, the number of labile protons on theresponsive CEST agents according to the invention can advantageously beincreased with consequent reduction of the amount of administered agent.

When compared to WO 02/43775, the present invention provides a generalmethod for the determination of a physical or chemical parameter ofdiagnostic interest in a human or animal body organ, fluid or tissue.The method is based on the use of a paramagnetic CEST contrast agentwhich is responsive for said parameter. According to this method, adetermination may be performed which is independent on the localconcentration of the administered agent.

The responsive paramagnetic agent for use in the method of the inventionpreferably includes at least one chelated complex of a paramagneticmetal ion. The paramagnetic metal ion is any transition or lanthanide(III) metal ion which has an electronic relaxation time suitably shortto significantly affect the chemical shift value of the mobile protonsto be irradiated. Preferred paramagnetic metal ions are selected in thegroup consisting of: iron (II) (high spin), iron (III), cobalt (II),copper (II), nickel (II), praseodymium (III), neodymium (III),dysprosium (III), erbium (III), terbium (III), holmium (III), thulium(III), ytterbium (III), and europium (III).

Lanthanide (III), also referred to as Ln(III), metal ions areparticularly preferred.

The chelating ligand of the paramagnetic complex for the use in themethod of the invention can be any organic ligand endowed with at leastone mobile proton bound to a nitrogen, oxygen, sulphur or phosphorousatom. Preferably, the mobile proton belongs to an amide groupcoordinated to the metal ion.

A further suitable source of mobile protons according to the inventionis represented by the water molecule(s) coordinated to the paramagneticcentre of the chelated complex. In this particular case the relaxationtime of the bulk water protons is influenced by the exchange thereofwith the inner-sphere coordinated water protons.

Responsive agents for use in the method of the invention includes thechelates of the macrocyclic tetra-amide derivatives of the1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), thetris-amide derivatives of the1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) and thederivatives of the hexa-aza-macrobicycle sarcophagine(3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane) with the preferred metalions indicated above.

Preferred paramagnetic contrast agents which include a chelating ligandof formula (I) are:

where: *R = R′ = R″ = R′″ = —CH₂—CONH—CH₂COOH Ligand A *R = R′ = R″ =R′″ = —CH₂—CONHNH₂ Ligand B *R = R′ = R″ = —CH₂—CONH₂

Ligand C *R = R′ = R″ = —CH₂—CONH₂

Ligand D *R = R′ = R″ = —CH₂—CONH₂

Ligand E *R = R′ = R″ = —CH₂—CONH—CH₂—COOBu

Ligand F *R = R′ = R″ = —CH₂—CONH₂

Ligand G *R = R′ = R″ = —CH₂—CONH₂

Ligand H *R = R′ = R″ = —CH₂—CONH—CH₂COOH

Ligand Lwhich is chelated to a Ln(III) metal ion, and which further preferablyincludes a water molecule coordinated to the paramagnetic metal centrethereof.

Another preferred class of compounds includes paramagnetic complexeshaving a chelating ligand of formula (II):

-   -   where:        -   R═R′═—NO₂ Ligand M

The 1,4,7,10-tetraazaciclododecane-1,4,7,10-acetic acid tetraazidederivative of formula

herein referred to as ligand B or DOTAM-hydrazide, as well as thechelated thereof with the transition or the lanthanide (III) metal ionsare new and are a further object of the present invention.

The 1,4,7,10-tetraazaciclododecane-1,4,7,10-acetic acidtetraglycineamide chelating ligand of formula:

is herein referred to as ligand A or DOTAM-Gly.

The CEST effect arising from the irradiation of the amide N—H protons ofthe Ln(III) complex compounds according to the invention has been testedon the basis of a series of CEST experiments in which the ST effect ismeasured as a function of the irradiation time (irradiation power of 25μT, where T means Tesla). In particular five Ln-DOTAM-Gly chelates inwhich Ln=Dy, Ho, Er, Tm, Yb were tested. The experimentation resultsincluded in FIG. 3 show that the most efficient saturation transfer isobserved for the Yb(III) complex (30 mM) at pH 8.1 wherein the observedsaturation effect of ca. 70% after 2 s of irradiation appears indicativeof a very efficient saturation transfer. To reach similar results withdiamagnetic molecules, such as aminoacids, a much higher concentration(125 mM) is requested. Furthermore, similar results are obtained only atpH 5, that is to say at pH values that are far from the physiologicalrange (Ward KM et al. J Magn Res 2000; 143: 79-87).

As far as other Ln(III)-DOTAM-Gly complexes are concerned, the datareported in FIG. 3 indicate a clear trend in the saturation transfereffect along the lanthanide series on passing from the Dy(III) chelate,for which the CEST effect is minimal, to the Yb(III) complex.

The overall visualization of the ST effects is represented by a CESTspectrum, an example of which is reported in FIG. 4 for a 30 mM solutionof Yb-DOTAM-Gly at pH 8.1. The spectrum reports the intensity of thewater signal, normalized to the higher value, as a function of theirradiation offset. Obviously, the effect is maximum at the resonancefrequency of water (0 ppm), but it is immediately evident that when theirradiation frequency is set to ca. −16 ppm from water, a significantwater saturation is observed and the residual signal is slightly lessthan 20%. However, since this effect may be partly accompanied by thedirect saturation of the water signal, the “true” CEST effect isquantitatively assessed by considering also the off-resonancesaturation. This means that the value of the M_(S)/M₀ ratio measured inthe experiment reported in FIG. 3 is the ratio of the two valuesindicated by the arrows in the FIG. 4.

For the Eu-DOTAM-Gly chelate the chemical shift separation between theamide protons and the bulk water is smaller (4.2 ppm) than the otherLn(III) ions tested (see FIG. 2). The irradiation of the amide protonswith the same square pulse used for the other Ln-DOTAM-Gly complexesdoes not allow the detection of a saturation transfer effect owing to aremarkable direct saturation effect on the bulk water. For this reason,it is convenient to use a selective shaped saturation pulse. The STeffect measured by using a train of 270° e-burp1 pulses of 20 ms each(total irradiation time 4 s, irradiation power 1.2 μT) was of 23% (pH7.7, 30 mM, 312° K, 7.05 T).

It is now noteworthy to consider that in this chelate it is alsopossible to detect a saturation transfer effect by irradiating the waterprotons coordinated to the Eu(III) ion which resonate at ca. 50 ppmdownfield from bulk water at 312° K. Since this signal is extremelybroad, it is again convenient to use a selective shape pulse in order toexcite all the spins simultaneously. On this basis, a remarkable STeffect of ca. 85% has been measured (train of 90° e-burp1 pulses of 1 mseach, total irradiation time 4 s, irradiation power 15.7 μT, pH 7.7, 30mM, 312° K, 7.05 T).

The ST effect shown by the lanthanide chelated complex according to theinvention is further markedly sensitive to physical or chemicalparameters of diagnostic interest wherein this allows their advantageoususe in the method of the invention for the determination of saidparameters either in vivo or in vitro.

A list of parameters of interest includes any physical or chemicalparameter of diagnostic interest which is able to influence at least onefactor which regulates the saturation transfer from the contrast agentand the surrounding water.

In particular said parameters include: temperature, pH, metaboliteconcentration, O₂ or CO₂ partial pressure, enzymatic activity, in ahuman or animal body organ or tissue.

According to the method of the invention the amount of saturationtransfer is related to the physical or chemical parameters of diagnosticinterest according to the following equation: $\begin{matrix}{\left( {1 - \frac{M_{s}}{M_{0}}} \right) = \left\lbrack {\frac{k_{ex}{n\lbrack C\rbrack}}{{2{R_{1{irr}}\left\lbrack {H_{2}O} \right\rbrack}} + {k_{ex}{n\lbrack C\rbrack}}}\left( {1 - {\exp\left\lbrack {{- \left( {R_{1{irr}} + \frac{k_{ex}{n\lbrack C\rbrack}}{2\left\lbrack {H_{2}O} \right\rbrack}} \right)}t} \right\rbrack}} \right)} \right\rbrack} & \lbrack 1\rbrack\end{matrix}$

According to this equation, the saturation transfer effect observed isconveniently quantified as (1−M_(S)/M₀) wherein Ms refers to theintensity of the water signal upon irradiation at the frequencycorresponding to the mobile protons resonance (v^(on)) and M⁰ indicatesthe water signal intensity measured upon irradiation at the frequencyv^(off) where v^(off)=−v^(on) and v^(water)=0.

The irradiation at v^(off) allows the evaluation of the directsaturation effects on the water signal.

As indicated by equation 1, the ST effect is dependent on:

-   -   the irradiation time, t;    -   the pseudo first order kinetic constant rate of the irradiated        protons k_(ex);    -   the number of irradiated mobile protons n;    -   the longitudinal relaxation rate of the bulk water upon        irradiation of the mobile protons, R_(lirr);    -   the molar concentration of the paramagnetic agent, [C] and of        the bulk water protons, [H₂O] (111.2 M in pure water).

One can safely assume that all the paramagnetic chelates preferred forthe use in the method of the invention possess an exchangeable watermolecule coordinated to the metal centre. For this reason, thoughR_(lirr) is conceptually different from R₁, it is likely that R_(lirr)is higher for paramagnetic systems than diamagnetic agent and,furthermore, it depends on both the concentration of the metal complexand the k_(ex) value.

Equation 1 indicates that the ST efficiency depends upon theconcentration of the exchanging protons and therefore on theconcentration of the contrast agent (n[C]). This finding makes themeasurement of the diagnostic parameter of interest dependent on thecontrast agent concentration. So, while no problems occur when thedetermination of said parameters is performed in vitro, where theconcentration may be determined, an accurate in vivo determinationthereof without knowing the local concentration of the administeredagent is not possible.

The relationship between the concentration of the paramagnetic agent andthe ST effect is not linear, unlike what is commonly observed for therelaxation enhancing ability of the conventional Gd(III)-based contrastagents. In fact, the steady-state value of the ST effect is determinedby: $\begin{matrix}{\left( {1 - \frac{M_{s}}{M_{0}}} \right) = \frac{k_{ex}{n\lbrack C\rbrack}}{{2{R_{1{irr}}\left\lbrack {H_{2}O} \right\rbrack}} + {k_{ex}{n\lbrack C\rbrack}}}} & \lbrack 2\rbrack\end{matrix}$where it is evident that the dependence of the concentration of thecontrast agent is less marked than in the conventional MRI contrastagents, even by taking into account the concentration dependence ofR_(irr).

So, in spite of the limited role played by the concentration on theefficacy of a CEST contrast agent, the precise knowledge of the localconcentration of the agent is still a necessary requisite for anaccurate determination in vivo of the parameter to be monitored.

This problem may be solved by considering two pools of magneticallydifferent labile protons whose ST effect shows a different dependencefrom the physico-chemical parameter of interest according to thepreferred method of the invention. To reach this aim, two strategies areindeed possible:

-   i) the use of a paramagnetic contrast agent comprising a single CEST    molecule endowed with both the two magnetically non equivalent    mobile protons pools, or-   ii) the use of a paramagnetic contrast agent comprising two    different CEST units endowed with at least one set of mobile protons    each. In the latter case the two molecules must have the same    biodistribution pattern.

In a first preferred method for the determination of physical orchemical parameters of diagnostic interest independently on the localcontrast agent concentration, a single paramagnetic compound having atleast two magnetically non equivalent pools of mobile protons inexchange with the bulk water protons is used.

According to this method, the selective irradiation is performed on thetwo different pools of mobile protons. A ratiometric method is thenexploited in order to remove the dependence of the ST effect from theabsolute concentration of the administered contrast agent and to allowthe determination of the physico-chemical parameter of interest both invitro (ex vivo) and in vivo independently from the local concentrationof the agent.

Preferably, the compound is a paramagnetic complex or a physiologicallyacceptable salt thereof. The metal ion is preferably selected amongparamagnetic transition or Ln(III) metal ions on the basis of theability to induce the ST effect through the involvement of twomagnetically different sets of mobile protons belonging to theparamagnetic complex molecule. The chelating ligand may consist of anyorganic monomeric ligand endowed with at least two pools or, if one poolis represented by the water protons coordinated to the metal centre ofthe paramagnetic complex, at least one pool of mobile protons bound to anitrogen, oxygen, sulphur, phosphorous atom. Preferably, the mobileprotons pools belong the first to an amide group of the chelating ligandand the second to the metal coordinated water protons. More preferably,the mobile protons pools belong the first to the coordinating amidegroups and the second to the metal bound water protons of the Eu(III)complexes of the chelating ligands A, C, D, E, F, G, H, L.

A different contrast agent for use in this preferred method includes aparamagnetic complex or a physiologically acceptable salt thereofwherein the chelating ligand consist of a dimeric ligand which includestwo equal or different chelating units each of them is endowed with atleast one pool of mobile protons and the two metal ions are suitablyselected among the transition or Ln(III) metal ions on the basis oftheir ability to induce a different ST effect through the involvement ofthe two sets of mobile protons pools. The mobile protons pools arepreferably represented: the first, by the water protons coordinated tothe metal ion of the first moiety, and the second by mobile protonsbound to a nitrogen, oxygen, sulphur, phosphorous atoms of the secondmoiety, or both of them may optionally be represented by the waterprotons coordinated to two different Ln (III) metal ions or by mobileprotons bound to a nitrogen, oxygen, sulphur or phosphorous atoms.

More preferably, the mobile protons pools belong, the first to the waterprotons coordinated to the Eu(III) metal ion and the second to the amidegroups coordinated to the Yb(III) of the dimeric complex compound[Eu—Yb(bisDOTAMGLy)] of formula

The (bisDOTAMGLy) dimeric chelating ligand as well as chelated complexesthereof with two transition or Ln(III) paramagnetic metal ions are newand constitute a further object of the present invention.

According to the second strategy, in another preferred method for thedetermination of physical or chemical parameters of diagnostic interestby use of CEST MRI imaging, two paramagnetic complex compounds are used.In this case, the two CEST agents must have the same biodistributionpattern. According to this method, the selective irradiation isperformed of two different pools of mobile protons, which are providedby the two paramagnetic agents. A ratiometric method is again exploitedwhich removes the dependence of the saturation transfer effect from theabsolute concentration of the administered contrast agents. This methodallows the determination of said physical or chemical parameter both invitro (ex vivo) and in vivo independently from the local concentrationof the agent but according to the known concentration ratio between thetwo agents.

Preferably, the two paramagnetic compounds are paramagnetic chelatecomplexes or a physiologically acceptable salt thereof in which the twochelated paramagnetic ions are different. Said two metal ions arepreferably selected among paramagnetic Ln(III) ions on the basis oftheir ability to promote the saturation transfer effect through theinvolvement of two magnetically different exchanging proton pools. Thetwo chelating ligands may consist of any organic ligand endowed with atleast one mobile proton bound to a nitrogen, oxygen, sulphur,phosphorous atom and may be equal or different but suitably selected inorder to grant the same biodistribution pattern for the two metalcomplexes. Preferably the mobile protons belong to an amide group and,more preferably, to the coordinating amide groups of a chelating ligandselected among: 1,4,7,10-tetraazaciclododecane-1,4,7,10-tetraacetic acidtetraglycineamide (ligand A),10-[(3-methoxyphenyl)methyl]-1,4,7,10-tetraazaciclododecane-1,4,7-tris-[(aminocarbonyl)methyl](ligand G) or to the metal coordinated water protons in thetetrahydrazide derivative (ligand B).

Even more preferred is the use in this method of a contrast agent whichcomprises Yb(III)-DOTAM-Gly together with Eu(III)-DOTAM-Gly or aphysiologically acceptable salt thereof. The first complex has beenchosen for the availability of chemically exchanging amide protons(Δδ=16 ppm at 312° K) and the second one for the protons belonging tometal coordinated water molecule (AB of ca. 50 ppm at 312° K).

Equally preferred is the use of Tm(III)-DOTAM-Gly (irradiation of amideprotons) together with Eu(III)-DOTAM-Gly or a physiologically acceptablesalt thereof.

Having the same electric charge, hydrophilic/lipophilic balance andanalogous structure, the two metal complexes of both the couples arereasonably expected to show the same biodistribution pattern.

In the most preferred method according to the invention for thedetermination of physical or chemical parameters of diagnostic interestindependently on the local contrast agent concentration a responsiveparamagnetic CEST contrast agent is administered endowed with at leasttwo different exchangeable proton or protons pools of which the first isonly responsive for the physical or chemical parameter of diagnosticinterest and the second shows a CEST effect which only depends on thelocal agent concentration.

According to this method, the selective irradiation is performed of thetwo different pools of mobile protons and the determination of thephysical or chemical parameter of interest both in vitro (ex vivo) andin vivo is obtained which is independent on the local contrast agentconcentration.

A further object of the invention is the use of a CEST contrast agentcomprising a paramagnetic chelate complex endowed with at least onemobile proton in chemical exchange with the water medium protons andable, when a proper radiofrequency rf irradiating field is applied atthe resonance frequency of said exchangeable proton, to generate asaturation transfer effect between said mobile proton and the waterprotons which is sensitive to a physical or chemical parameter ofdiagnostic interest for the preparation of a pharmaceutical compositionfor the determination of said parameter in a human or animal body organ,fluid or tissue by use of CEST MR Imaging.

Preferably said paramagnetic compound(s) include a chelating ligand offormula (I)

where: *R = R′ = R″ = R′″ = —CH₂—CONH—CH₂COOH Ligand A *R = R′ = R″ =R′″ = —CH₂—CONHNH₂ Ligand B *R = R′ = R″ = —CH₂—CONH₂

Ligand C *R = R′ = R″ = —CH₂—CONH₂

Ligand D *R = R′ = R″ = —CH₂—CONH₂

Ligand E *R = R′ = R″ = —CH₂—CONH—CH₂—COOBu

Ligand F *R = R′ = R″ = —CH₂—CONH₂

Ligand G *R = R′ = R″ = —CH₂—CONH₂

Ligand H *R = R′ = R″ = —CH₂—CONH—CH₂COOH

Ligand Lwhich is chelated to a Ln(III) metal ion.

Another preferred paramagnetic compound(s) include a chelating ligand offormula (II)

where:*R=R′=—NO₂  Ligand M

Even preferably said paramagnetic compound is the [Eu—Yb(bisDOTAMGLy)]chelated complex.

In an even further aspect the invention relates to the use of twoparamagnetic chelated complex which must exhibit the samebiodistribution pattern, each of them comprising at least one mobileproton in chemical exchange with the water medium protons for thepreparation of a pharmaceutical composition for the determination of aphysical or chemical parameter of diagnostic interest in a human oranimal body organ, fluid or tissue independently on the local contrastagent concentration, by use of CEST MR Imaging. Preferred is the use forthis scope of Yb(III)-DOTAM-Gly together with Eu(III)-DOTAM-Gly or aphysiologically acceptable salt thereof or, optionally,Tm(III)-DOTAM-Gly together with Eu(III)-DOTAM-Gly.

In an even further aspect the invention relates to a pharmaceuticalcomposition which includes, together with a physiologically tolerablecarrier, a CEST contrast agent comprising a paramagnetic chelatecomplex, or a physiologically acceptable salt thereof, which comprisesat least one, and preferably at least two, mobile protons or protonpools in chemical exchange with the water medium protons able, when aproper radiofrequency rf irradiating field is applied at the resonancefrequency of the said exchangeable protons, to generate a saturationtransfer effect between said mobile proton and the water protons whichis only sensitive to the physical or chemical parameter of diagnosticinterest.

This pharmaceutical composition preferably includes, together with aphysiologically tolerable carrier, a paramagnetic chelated complexselected among: Eu(III)-DOTAM-Gly, Eu(III)-DOTAM-hydrazide, Co(II)(highspin) chelate complex of the ligands G and M, the Ln(III) chelatecomplexes of the chelating ligands C, G, H and L, [Eu—Yb(bisDOTAMGLy)]or a physiologically acceptable salt thereof.

Also preferably, the pharmaceutical composition includes, together witha physiologically tolerable carrier, two paramagnetic chelated complexesor a physiologically acceptable salt thereof wherein the twoparamagnetic metal ions are different and the chelating ligands, whichmay consist of any organic ligand endowed with at least one mobileproton bound to a nitrogen, oxygen, sulphur, phosphorous atom, aresuitably selected in order to grant the same biodistribution pattern forthe two metal complexes. More preferably the chelating ligand are boththe same.

The two paramagnetic complexes are preferably comprised in equal molaramount or in a known molar ratio which is selected according to thechelated metal ions. Said ratio may range from 1 to 30, preferably itranges from 1 to 10, more preferably from 1 to 5 and most preferablyfrom 1 to 2, wherein the minimum molar concentration requested of theparamagnetic compound included in lower amount is at least 0.05 mM whilethe global concentration of the included paramagnetic CEST contrastagent ranges between 0.001 and 1.0 M.

Most preferably, this pharmaceutical composition comprises Yb(III)DOTAM-Gly together with Eu(III) DOTAM-Gly or Tm-DOTAM-Gly together withEu-DOTAM-Gly, or a physiologically acceptable salt thereof, togetherwith a physiologically acceptable carrier.

The pharmaceutical preparations according to the invention can besuitably injected intravasally (for instance intravenously,intraarterially, intraventricularly, and so on) or used by way ofintrathecal, intraperitoneal, intralymphatic, intracavital, oral orenteral administration.

The injectable pharmaceutical formulations are typically prepared bydissolving the active ingredient(s) and the pharmaceutically acceptableexcipients in water of suitable purity from the pharmacological point ofview. The resulting formulation is suitably sterilised and can be use assuch or it can alternatively be lyophilised and reconstructed before theuse.

These formulations can be administered in concentrations depending onthe diagnostic requirements, at a dose ranging from 0.01 to 0.5 mmol/kgbody weight.

In order to test the validity of a preferred method according to theinvention the CEST spectra of a solution containing 16 mM ofEu-DOTAM-Gly and 20 mM of Yb-DOTAM-Gly at pH 8.1 (7.05 T, 312° K,irradiation power 25 μT, irradiation time 4 s) were registered and theresults are included in FIG. 8. Interestingly, the detection of a “peak”(very broad) centred at about 50 ppm (downfield the water signal) is aclear indication of the saturation transfer occurring when thecoordinated water protons of Eu-DOTAM-Gly are irradiated.

The ratiometric method on which is based the preferred method accordingto the invention is derived from a re-arrangement of equation 1 and itis expressed by the following equation: $\begin{matrix}{\frac{\left( \frac{M_{0} - M_{s}}{M_{s}} \right)^{A}}{\left( \frac{M_{0} - M_{s}}{M_{s}} \right)^{B}} = \frac{K^{conc}k_{ex}^{A}n^{A}R_{1{irr}}^{B}}{k_{ex}^{B}n^{B}R_{1{irr}}^{A}}} & \lbrack 3\rbrack\end{matrix}$where the superscripts A and B identify the paramagnetic complexcompounds whose exchanging pools magnetic parameters are referred to. Inthe above experimentation, for example, A=Yb-DOTAM-Gly andB=Eu-DOTAM-Gly and K^(conc) which represents the [A]/[B] ratio is 1.25.The presence of two R_(lirr) values, one for each pool of labile protonirradiated, is due to the fact that, in principle, R_(lirr) depends onthe exchange rate between the bulk water and the irradiated mobileprotons which is different for the two proton pools.

The same equation holds also if one single compound with two pools ofmobile protons is considered, but, in this case, K^(conc) is obviouslyequal to 1.

pH Responsive CEST Agents

Generally speaking, a good candidate as pH responsive CEST agentaccording to the method of the invention may be any paramagnetic complexcompound which includes a Ln(III) metal ion or a transition metal and achelating ligand which comprises at least one mobile proton whosechemical exchange with the water protons undergoes a basic or acidcatalysis.

Moreover, any paramagnetic complex whose structure changes according tothe pH in such a way to induce a chemical shift modification of themobile proton(s) thereof can equally be used as pH responsive CEST agentaccording to the method of the invention.

Suitable pH responsive CEST agents further include all the paramagneticcomplex compounds wherein the number of water molecules coordinated tothe paramagnetic metal centre changes depending on the pH and a changeof the relaxation rate of the bulk water protons occurs. A pH responsiveagent of this kind is represented, for example, by a Ln(III) chelatedcomplex of the chelating Ligand H of formula

When the sulphonamide group is in the protonated form, the Ln(III) ionis nona-coordinated and two mobile water molecules are bound to themetal centre. On the contrary, when the sulphonamide group isdeprotonated it becomes able to coordinate the metal ion which result tobe octa-coordinated (no water molecules coordinated to it). The chemicalshift of the 6 exchangeable amide protons on the three pendent armsdepends on the molar ratio between the protonated and the deprotonatedform of the complex, that is to say with the pH of the solution. In thisway the saturation transfer effect upon irradiation of the amide protonsof the complex (in the protonated or in the deprotonated form) issensitive to the pH of the surrounding medium.

A further example of pH responsive paramagnetic CEST agent isrepresented by the Eu-DOTAM-hydrazide (ligand B) chelated complex. Inthis compound a proton dissociation, occurring at pH above 6.5, causes asignificant acceleration of the exchange rate of the water moleculebound to Eu(III) ion on passing from the condition of slow exchange(k_(ex)Δv<1/2π) at pH<6.5 to the coalescence (k_(ex)Δv>1/2π) between thetwo signals. Indeed, the ST observed upon irradiation at the resonancefrequency of the water protons bound to the Eu(III) ion (Δv of 15000 Hz)decreases at pH values higher than 6.5 (FIG. 5).

As the pH dependence of the ST effect promoted by the mobile protons ofamide groups thereon, the Ln(III) DOTAM-Gly and the Ln(III)-Ligand Gcomplexes represent an example of suitable class of pH responsive CESTagents.

The pH dependence of the ST effect has been assessed for the Yb-Ligand Gcomplex compound (20 mM, 312° K, irradiation power 25 μT, irradiationtime 4 s) and the results are showed in FIG. 6. The ST effect ismarkedly pH-dependent, being maximum at pH 7.2 and almost negligible atpH values lower than 5.5.

The pH dependence of the ST effect has been also assessed for theYb-DOTAM-Gly derivative (30 mM, 312° K, irradiation power 25 μT,irradiation time 4 s) and the results are showed in FIG. 6 Bis. The STeffect is markedly pH-dependent, being maximum at pH 8.1 and almostnegligible at pH lower than 6. The pH dependence is linear (regressioncoefficient=0.996) in the pH range 5.5-8.1, whereas at higher pH valuesthe saturation transfer becomes less efficient likely because of the tooextensive exchange broadening of the N—H resonances. This behavioursupports the hypothesis that the pH dependence of the ST effect mainlyarises from the base-catalysed proton exchange of the amide N—H protonsof the Yb(III) complex. Interestingly, similar results were obtainedupon 2 s of irradiation. These results are very promising for an in vivoapplication of this chelate, since the ST effect is markedly pHsensitive and, moreover, it is properly tuned at the physio-pathologicalpH interval.

A standard proton density (PDW) ¹H water MR image of a phantomcontaining a 30 mM solution of the agent at different pH values wasfurther recorded at 7.05 T and 298° K on a Bruker Pharmascan imager(FIG. 7). Interestingly, even at pH 5.4, where the directly measuredCEST effect is quite low (12%), the corresponding contrast in the imagedifference is not negligible at all.

According to the preferred method of the invention, the pH dependence ofthe CEST effect has been tested by using a mixture of twoLn(III)-DOTAM-Gly chelates differing in the lanthanide ion. Thus,Yb-DOTAM-Gly and Eu-DOTAM-Gly complexes were chosen in order to exploitthe CEST effects associated with the exchange of the amide N—H protonsand the metal coordinated water protons, respectively. In FIG. 8 theCEST spectrum obtained from a solution containing 16 mM of Eu-DOTAM-Glyand 20 mM of YbDOTAM-Gly at pH 8.1 (B₀=7.05 T, 312° K, irradiation power25 μT, irradiation time 4 s) is shown. Besides the peak due to thedirect saturation of the bulk water, the CEST spectrum is characterizedby two additional peaks: one, relatively narrow, is upfield shifted ofca. 16 ppm and the other, very broad, is downfield shifted at ca. 50 ppmfrom the chemical shift of the bulk water. Clearly, the first peakcorresponds to the four amide N—H protons of the Yb(III) complex,whereas the second peak refers to the protons of the coordinated waterin the Eu(III)-based chelate. According to the ratiometric method onwhich the method of the invention is based, the CEST effect evaluated as[(M₀−M_(S))/M_(S)]_(YbL)/[(M₀−M_(S))/M_(S)]_(EuL) ratio is not dependenton the absolute concentration of the contrast agents but only on theirrelative concentration ratio. On this basis, the pH dependence of the[(M₀−M_(S))/M_(S)]_(YbL)/[(M₀−M_(S))/M_(S)]_(EuL) ratio was investigatedat 7.05 T, 312 K on a solution containing 16 mM of Eu-DOTAM-Gly and 20mM of the Yb(III)-based chelate (irradiation time 4 s, irradiation power25 μT). The result reported in FIG. 9 shows the high responsiveness tothe pH of the system of the invention. Interestingly, the marked pHdependence observed for such system, which ensures a good sensitivity tothe method, is basically due to the pH dependence of the ST effect shownby the Yb(III) chelate. In fact, the ST effect arising from theirradiation of the protons of the coordinated water in the Eu-DOTAM-Glycomplex is basically unaffected in the investigated pH range from 5.5 to8.5 (FIG. 10). This allows the full exploitation of the pH dependence ofthe Yb(III) chelate which results in a remarkable pH dependence of theST ratio in pH range from 6.5 to 8.1 considerably larger than the onereported by Balaban and Ward in their diamagnetic system (Ward KM andBalaban RS. Magn Res Med 2000; 44: 799-802).

In another experiment we have checked the validity of one of thepreferred method of the invention for the pH determination in vitro whena single paramagnetic complex endowed with two magnetically nonequivalent pools of mobile protons is used. In FIG. 11 the pH dependenceof the ratiometric plot (amide protons over coordinated water protons)for a 30 mM solution of Eu-DOTAM-Gly at 312° K and 7.05 T is reported.

The experiment has been carried out by irradiating the two pools ofmobile protons for 2 seconds with the same modality of irradiation(selective e-burp pulses) reported above for this complex.

The data reported in FIG. 11 suggest that the pH dependence ismaintained, even if the sensitivity of this method is quitesignificantly reduced with respect to the data reported in FIG. 9. Thereason is mainly due to the lower efficiency of the ST effect for theamide protons of Eu(III)-DOTAM-Gly complex.

The pH dependence of the ST effect has been also assessed for the[Yb-Eu(bisDOTAM-Gly)] derivative (30 mM, 312 K, irradiation power 25 μT,irradiation time 4 s) and the result, under the form of the ratiometricplot (amide protons of Yb(III) versus metal bound water protons ofEu(III)), is showed in FIG. 12. The sensitivity of the ratiometric plotto the pH of the solution for the dimer is lower than the mixture ofEu(III) and Yb(III)-DOTAMGly (FIG. 9). It is likely that this differenceis due to the lower K^(conc) value (1 vs 1.25) for the dimer.Nevertheless, the sensitivity of the dimer is higher than that oneobserved when the single Eu(III)-DOTAMGly complex (FIG. 11) is used.

Temperature Responsive CEST Agents

The exchange rate of any mobile proton is temperature dependent.

The temperature can also affect the chemical shift value of theexchanging protons induced by the paramagnetic metal and the T₁ value ofthe water signal. On this basis, any paramagnetic complex whosechelating ligand includes at least a mobile proton can advantageously beused as temperature responsive CEST agent according to the method of theinvention.

In the case of mixed valence compounds (D. E. Richardson and H. Taube inCoord. Chem. Rev. 1984, 60:107-129), the change of the electron spinconfiguration caused by a temperature variation can also be exploitedfor the attainment of a very efficient temperature responsive CEST agentaccording to the method of the invention.

The responsiveness to the temperature according to the method of theinvention has been tested for the Eu-DOTAM-hydrazide complex compound(30 mM; pH 7.4). The temperature dependence of the saturation transferhas been tested by irradiating the mobile water protons coordinated tothe metal centre. The result is reported in FIG. 13. The responsivenessto the temperature according to the method of the invention has beentested also by use of a composition containing Tm(III)-DOTAM-Glytogether with the Eu(III) complex of the same ligand. Upon irradiationof the amide protons of the Tm(III)-DOTAM-Gly and the water protonsmetal coordinated to the Eu(III)-complex, according to the method of theinvention, a satisfactory results has been obtained, as shown in FIG. 13Bis.

CEST Agents Responsive to the Concentration of Metabolites

In order to be responsive to a specific metabolite, a paramagnetic CESTagent has to be able to interact non covalently and as selectively aspossible with it and this interaction must promote changes in theparameters determining the saturation transfer efficacy such as, forexample, chemical shift, exchange rate, relaxation rate of bulk water,number of mobile protons.

The responsiveness to a given metabolite according to a preferred methodof the invention has been tested in vitro by using the Yb(III) complexof the heptadentate10-[(3-methoxyphenyl)methyl]-1,4,7,10-tetraazaciclododecane-1,4,7-tris-[(aminocarbonyl)methyl]chelating ligand (Ligand G). The chelating ligand of this paramagneticcomplex has been prepared as disclosed in the European patentApplication 01124440.7. This chelate owns 6 mobile amide protons whosechemical shift separation from the bulk water is ca 29 ppm upfield thebulk water signal at 312° K. This complex is able to interact quitestrongly with several anionic substrate endowed with coordinating groupsable to replace the water molecules coordinated to the metal centre ofthe chelate complex.

Among the anionic substrates of interest for this application, one mayinclude both endogenous and exogenous compounds.

More preferably, the endogenous substrates are selected from the groupconsisting of lactate, citrate, carbonate, phosphate, pyruvate, naturalamino-acids, oxalate, tartrate, succinate, choline, creatine, acetate,and malonate.

Even more preferably, the substrates are human metabolites, whereinlactate, citrate, carbonate, and phosphate are the most preferred.

Moreover, the substrate molecule of interest for the method of theinvention can be an exogenous substance, wherein the term exogenous, asused herein, refers to any substance of pharmacological or diagnosticinterest, eventually modified in order to allow a suitable binding tothe paramagnetic complex.

As a representative, but not limiting, example we have consideredL-lactate.

The affinity constant between the metal complex and L-lactate has beenevaluated through relaxometric measurements carried out on the Gd(III)complex (K_(A) of ca. 3000 at 298° K and pH 6.5). The exchange betweenthe free and the lactate-bound Yb(III) complex is slow on the NMRfrequency timescale. Therefore, different resonances for the mobileamide protons for the two forms (Δω of 29 ppm and 15 ppm for the free-and bound-forms, respectively) of the metal complex may be detected atphysiological conditions (312° K and pH 7.4) in the ¹H-NMR spectrum.

The signals corresponding to the two forms of the CEST agent aresufficiently separated to allow their selective irradiation. Thedependence on the L-lactate concentration of the ST effect promoted bythe irradiation of the free amide protons in a 9.3 mM solution ofYb(III) complex of the heptadentate0.10-[(3-methoxyphenyl)methyl]-1,4,7,10-tetraazaciclododecane-1,4,7-tris-[(aminocarbonyl)methyl]chelating ligand is shown in FIG. 14 (7.05 T, pH 7.4, 312 K, irr. power1050 Hz, irr. time 6 s).

Interestingly, the ST efficiency shows a marked dependence in the rangeof Lactate concentration (0-10 mM) of diagnostic relevance.

This result has been confirmed by recording a PDW ¹H-MR image (298 K, pH7.4, 7.05 T) of a phantom consisting of solutions at differentconcentrations of L-lactate in the 0-10 mM range (FIG. 15).

The independence of the ST effect on the concentration of the CEST agentcan be achieved, according to this invention, by use, for example, of adimeric complex compound comprising Yb-G and Eu-DOTAM-Gly units.

CEST Agents Responsive to the Partial Pressure of O₂

The Co(II) high spin chelated complex of the ligand G represent anexample of a paramagnetic CEST agent responsive to partial pressure ofthe O₂ for use in the method of the invention.

According to the following scheme:

O₂ can transform the Co(II)-high spin complex into the Co(III)-low spindiamagnetic complex wherein this results in a considerable reduction ofthe CEST effect upon irradiation of the exchangeable amide protonscoordinated to the metal centre of the complex compound.

As another example of CEST agents responsive to the redox potential ofthe medium, Ln(III) complexes of Ligand L have been prepared.

These ligands contains a diphenylamine substituent which act as aredox-switch whose redox potential is very close to that one present invivo (ca. 0.8 V).

A third example of CEST agents sensitive to the partial pressure of O₂is represented by the Co complex of Ligand M. Analogously to the Co-Gcomplex, the responsiveness of this system is due to the redoxequilibrium between the paramagnetic high spin Co(II) form and thediamagnetic low-spin Co(III) compound. Unlike from the other examples,here the ST effect is measured upon irradiation of the amine protons ofthe complexes.

The dependence of the ST effect on the partial pressure of O₂ can beobserved upon irradiating of either the frequency resonance of the metalbound water protons in the Eu(III) complex or the amide protons of theother Ln(III) chelates. The concentration independence of the ST effectcan be obtained, according to this invention, by using a single complex(Eu(III)-L chelate), a mixture of Eu(III)-L and Yb(III)-L compounds orby using a dimer constituted by the redox-sensitive unit (e.g.Yb(III)-L, Co-G or Co-M complexes) linked to a DOTAM-Gly unit containingEu(III) ion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: ¹H-NMR spectra of Yb-DOTAM-Gly (7.05 T, 298 K and pH 7) in D₂O(top) and H₂O (bottom).

FIG. 2: Chemical shift difference (Δω in ppm) between the amide N—Hprotons and bulk water for Ln-DOTAM-Gly chelates. (7.05 T, 312 K)

FIG. 3: Dependence of the saturation transfer on the irradiation time(irradiation power 25 μT) for Dy-(30 mM, down-triangle), Ho-(30 mM,up-triangle), Er-(40 mM, diamond), Tm-(40 mM, circle) and Yb-(30 mM,square) chelates of DOTAM-Gly (B₀=7.05 T, 312 K, pH 8.1).

FIG. 4: CEST spectrum of a 30 mM solution of Yb-DOTAM-Gly at pH 8.1(B₀=7.05 T, 312 K, irradiation time 4 s, irradiation power 25 μT).

FIG. 5: pH dependence resulting by the irradiation of the mobile waterprotons coordinated to Eu-DOTAM-hydrazide (30 mM; pH 7.4, B₀=7.05 T, 312K, irradiation time 2 s, irradiation power 15.7 μT).

FIG. 6: pH dependence of the saturation transfer effect for a 20 mMsolution of Yb-ligand G (B₀=7.05 T, 312 K, irradiation power 25 μT,irradiation time 4 s).

FIG. 6 Bis: pH dependence of the saturation transfer effect for a 30 mMsolution of Yb-DOTAM-Gly (B₀=7.05 T, 312 K, irradiation power 25 μT,irradiation time 4 s).

FIG. 7: 7.05 T PDW Spin-echo image of a phantom containing 4 vials ofYb-DOTAM-Gly (30 mM) in the pH range 5.4-8.4. The vials were dipped inwater containing 30 mM of Yb(III) aqua-ion (298 K, irradiation time 4s). The image is the difference between two PDW images (TR=3.04 s;TE=18.3 ms) in which the pre-saturation pulse was centred first on theamide protons (−4794 Hz from bulk water) and then symmetricallyoff-resonance (4794 Hz from bulk water).

FIG. 8: CEST spectra of a solution containing 16 mM of Eu-DOTAM-Gly and20 mM of Yb-DOTAM-Gly at pH 8.1 (B₀=7.05 T, 312 K, irradiation time 4 s,irradiation power 25 μT).

FIG. 9: pH dependence resulting by the exploitation of the ratiometricmethod (Eu-DOTAM-Gly concentration 10 mM, Yb-DOTAM-Gly concentration12.5 mM; 7.05 T, 312 K, irradiation time 4 s, irradiation power 25 μT).The error bars indicate the standard deviation of 5 independentmeasurements.

FIG. 10: pH dependence of ST effect for a solution containing 10 mM ofEu-DOTAM-Gly and 12.5 mM of Yb-DOTAM-Gly (7.05 T, 312 K, irradiationtime 4 s, irradiation power 25 μT). The square refer to the irradiationof the amide protons of the Yb(III) complex and the circle correspond tothe irradiation of the coordinated water protons in the Eu(III) chelate.

FIG. 11: pH dependence resulting by the exploitation of the ratiometricmethod of a 30 mM solution of Eu-DOTAM-Gly (7.05 T, 312 K, irradiationtime 2 s). The saturation has been performed by using a train of 270°e-burp1 pulse (20 ms each, power 1.2 μT) for the amide protons and atrain of 90° e-burp1 pulse (1 ms each, power 15.7 μT) for the metalbound water protons.

FIG. 12: pH dependence resulting by the exploitation of the ratiometricmethod for the Yb—Eu-bisDOTAM-Gly dimer (agent concentration 30 mM,B₀=7.05 T, 312 K, irradiation time 4 s, irradiation power 25 μT).

FIG. 13: Temperature dependence resulting by the irradiation of themobile water protons coordinated to Eu-DOTAM-hydrazide (30 mM; pH 7.4,B₀=7.05 T, 312 K, irradiation time 2 s, irradiation power 15.7 μT).).

FIG. 13 Bis: Temperature dependence resulting by the exploitation of theratiometric method (Eu-DOTAM-Gly=14 mM; Tm-DOTAM-Gly=14 mM; 7.05 T, pH7.4, irradiation time 4 s, irradiation power 25 μT).

FIG. 14: Dependence on the L-lactate concentration of the ST effect of a9.3 mM solution of Yb(III) complex of the10-[(3-methoxyphenyl)methyl]-1,4,7,10-tetraazaciclododecane-1,4,7-tris-[(aminocarbonyl)methyl]chelating ligand measured by irradiating the amide protons of the freechelate at −29 ppm from the bulk water protons (7.05 T, 312 K, pH 7.4,irr. time 4 s, irr. power 1050 Hz).

FIG. 15: 7.05 T PDW Spin-echo image of a phantom containing 4 vials ofYb-Ligand G (9.3 mM) containing different L-lactate concentrations inthe 0-10 mM range. The image is the difference between two PDW images inwhich the saturation pulse was centred first on the amide protons of thefree complex (−8700 Hz from bulk water) and then symmetricallyoff-resonance (8700 Hz from bulk water).

EXPERIMENTAL SECTION

The preparation of the compounds of the invention has been performedaccording to procedures and synthesis steps well known to a man skilledin the art. Non limiting examples are included below.

Preparation of 1,4,7,10-tetraazaciclododecane-1,4,7,10-acetic acidtetraglycineamide (DOTAM-Gly)

The chelating ligand was synthesized according to the following steps:

-   a) exhaustive alkylation of TAZA    (TAZA=1,3,5,7-tetraazacyclododecane, 0.075 mol) with    N-(2-Bromoethanoyl) ethyl glycinate (0.3 mol) in the presence of 0.3    mol of K₂CO₃ as base to give the corresponding tetraethyl ester    derivative.

The reaction was carried out in acetonitrile by heating at 70° C. for 6h. After removal of the undissolved materials by filtration, the productwas simply obtained by evaporating the solvent. Yield: 91.4%

N-(2-Bromoethanoyl) ethyl glycinate was syntetisized according to thepublished procedure. (Kataki R, et al. J Chem Soc Perkin Trans 2 1992;8:1347-1351).

-   b) controlled saponification of the tetraethyl ester and isolation    of the desired tetracarboxylic acid.

The saponification of the tetraester was carried out in 200 mL ofethanol/water (1:1) solution at 60° C. NaOH 1 N (232 mL) was added tomaintain the pH of the solution constant (pH 11) for almost 45′. Thereaction was complete after 1 h heating. The resulting orange solutionwas cooled down and acidified at pH 2.2 with HCl. The DOTAM-Gly ligandwas separated from such solution by liquid chromatography (solid-phase:Amberlite® XAD-1600; eluent: water). Yield: 88%.

The ligand has been characterized by MALDI-TOF Mass Spectrometry (calc.for C₃₂H₄₀N₈O₁₂, 632.63 amu; found 633.55 (MH+)).

Synthesis of the Ln(III) Complexes

The Ln(III)-DOTAM-Gly complexes were prepared by mixing equimolar amount(0.3 mmol) of the ligand and the corresponding Ln(III) chloride in 10 mLof water (room temperature, pH 8, 30′). The recovered chelate complexeshave been characterized by means of their ¹H-NMR spectra. The recovereddata were consistent with the expected structures.

Preparation of the Ligand B (DOTAM-Gly).

The synthesis has been carried out through the following steps:

-   -   Synthesis of N-(Benzyl-oxy carbonyl)-N′-(bromoacetyl)hydrazine        (dichloro-methane, 0° C.)    -   Synthesis of        1,4,7,10-tetra{2-[N′-(benzyl-oxy-carbonyl)hydrazino]-2-oxo-ethyl}-1,4,7,10-tetraazacyclododecane        (acetonitrile, room temperature):    -   Synthesis of ligand B (methanol, room temperature)

Preparation of the Ligand C

Main steps include:

-   -   Synthesis of 1-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane        (acetonitrile/water 10:1, 60° C.)    -   Synthesis of        1-(4-nitrophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetamide        (acetonitrile, room temperature)

Preparation of the Dimeric Chelated Complex [Eu—Yb(bisDOTAMGLy)]

The global preparation has been performed according to a procedureschematized below:

Those skilled in the art should realize that, by use of the above schemeand by suitably changing the chelating unit or the chelated metal ionany dimeric contrast agent according to the method of the invention canbe easily obtained.

NMR Methods

The high resolution work has been carried out on a Bruker Avance 300spectrometer operating at 7.05 T.

The saturation transfer experiments were carried out at 312° K byirradiating the sample with a continuous wave presaturation square pulse(power of 1050 Hz) or by using a proper train of e-burp1 selectivepulses. Four scans and 4 dummy scans were used for all the experiments.

NMR imaging was performed using a 7.05 T Bruker PharmaScan havingactively shielded gradients 300 mT/m) and running ParaVison 2.1.1software. Standard PDW (proton density weighted images) were obtainedusing a SE (spin-echo) imaging sequence (using Hermite shaped 90° and180° pulses). NMR image adopted parameters were (TR/TE/NE=3.0 s/18.3ms/l); FOV (Field Of View) 30×30 mm²; slice thickness 2 mm and imagematrix 256×256 points. A 2.25 Watt square shaped saturation pulse wasapplied for 4 s in the pre-delay of the spin-echo sequence. Two imageswere acquired, one with saturation of the amide protons at −4794 Hz frombulk water protons and the other with the rf irradiation offset at 4794Hz.

1) A method for the determination, by use of the CEST MRI technique, ofa physical or chemical parameter in a human or animal body organ, fluidor tissue, wherein: a responsive paramagnetic CEST contrast agent isemployed comprising at least one exchangeable proton whose saturationcapability is correlated to the physical or chemical parameter ofinterest, and a CEST MR image responsive for said parameter isregistered. 2) The method of claim 1 wherein the CEST contrast agentcomprises at least two magnetically non equivalent pools of mobileprotons. 3) The method of claim 1 in which the determination isperformed in vitro or ex vivo. 4) The method of claim 2 wherein thedetermination is performed in vivo. 5) The method of claim 1 wherein theparameter of interest is selected from temperature, pH, metaboliteconcentration, O₂ or CO₂ partial pressure and enzymatic activity. 6) Themethod of claims 1 and 2 wherein the responsive paramagnetic CESTcontrast agent comprises a paramagnetic chelate compound in which thechelating ligand is an organic ligand containing at least one mobileproton bound to a nitrogen, oxygen, sulphur or phosphorous atom and theparamagnetic ion is selected from the group consisting of iron (II)(high spin), iron (III), cobalt (II), copper (II), nickel (II),praseodymium(III), neodimium (III), dysprosium (III), erbium (III),terbium (III), holmium (III), thulium (III), ytterbium (III), andeuropium (III), or a physiologically acceptable salt thereof. 7) Themethod of claim 6 wherein the paramagnetic chelate complex comprises awater molecule coordinated to the paramagnetic centre. 8) The method ofclaim 7 wherein the mobile proton in the CEST contrast agent eitherbelong to an amide group of the chelating ligand or is a metal boundwater proton of said paramagnetic chelate complex. 9) The method ofclaims from 6 to 8 wherein the chelating ligand is a tetra-amidederivative of the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTA), or the tris-amide derivatives of the1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), or aderivative of the hexa-aza-macrobicycle sarcophagine(3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane). 10) The method of claim2 wherein the CEST contrast agent comprises a single a paramagneticcomplex compound endowed with both the two magnetically non equivalentpools of mobile protons or a physiologically acceptable salt thereof.11) The method of claim 2 wherein the CEST contrast agent comprises twoparamagnetic complex compounds which have the same biodistributionpattern, each of them providing a different pool of mobile protons. 12)The method of claim 9 wherein the chelating ligand is a compound offormula (I)

where: *R = R′ = R″ = R′″ = —CH₂—CONH—CH₂COOH Ligand A *R = R′ = R″ =R′″ = —CH₂—CONHNH₂ Ligand B *R = R′ = R″ = —CH₂—CONH₂

Ligand C *R = R′ = R″ = —CH₂—CONH₂

Ligand D *R = R′ = R″ = —CH₂—CONH₂

Ligand E *R = R′ = R″ = —CH₂—CONH—CH₂—COOBu

Ligand F *R = R′ = R″ = —CH₂—CONH₂

Ligand G *R = R′ = R″ = —CH₂—CONH₂

Ligand H *R = R′ = R″ = —CH₂—CONH—CH₂COOH

Ligand L

or a compound of formula (II):

where: R═R′═—NO₂ 13) The method of claim 10 wherein the mobile protonsbelong the first to an amide group of a monomeric chelating ligand andthe second to a metal coordinated water molecule. 14) The method ofclaim 13 wherein the mobile protons belong the first to the coordinatingamide groups and the second to the metal bound water protons of theEu(III) complexes of the chelating ligands A, C, D, E, F, G, H, L. 15)The method of claim 10 wherein the single paramagnetic complex compoundcomprises a dimeric chelating ligand which includes two chelating units,which are the same or different, chelated with two different Ln(III)metal ions. 16) The method of claim 15 wherein the chelate complex is[Eu—Yb(bisDOTAMGLy)]. 17) The method of claim 11 wherein the CESTcontrast agent comprises Eu(III) DOTAM-Gly optionally together withYb(III) DOTAM-Gly or Tm(III)-DOTAM-Gly. 18) A pharmaceutical compositionwhich includes, together with a physiologically tolerable carrier, aCEST contrast agent comprising a paramagnetic chelated complex, or aphysiologically acceptable salt thereof, which comprises at least onemobile proton in chemical exchange with the water medium protons andwhich is able, when a proper radiofrequency rf irradiating field isapplied at the resonance frequency of the said exchangeable proton, togenerate a saturation transfer effect between said mobile proton and thewater protons which is only sensitive to the physical or chemicalparameter of diagnostic interest. 19) The pharmaceutical composition ofclaim 18 wherein the CEST contrast agent contains a paramagneticchelated complex comprising at least two magnetically non equivalentpools of mobile protons or two paramagnetic chelated complexes havingthe same biodistribution pattern each of them providing a different poolof mobile protons, or a physiologically acceptable salt thereof. 20) Thepharmaceutical composition of claim 19 comprising two paramagneticcomplexes in a molar ratio ranging from 1 to
 30. 21) The pharmaceuticalcomposition of claim 20 wherein the two paramagnetic complexes are in amolar ratio ranging from 1 to
 5. 22) The pharmaceutical composition ofclaims 19 to 21 which comprises Eu(III)-DOTAM-Gly optionally togetherwith Yb-DOTAM-Gly or Tm-DOTAM-Gly. 23) The use of a CEST paramagneticcontrast agent comprising a paramagnetic chelating complex endowed withat least one mobile proton in chemical exchange with the water mediumprotons and able, when a proper radiofrequency rf is applied at theresonance frequency of said exchangeable proton, to generate asaturation transfer effect between said mobile proton and the waterprotons which relates to a physical or chemical parameter of diagnosticinterest for the preparation of a pharmaceutical composition for thedetermination of said parameter in a human or animal body organ, fluidor tissue by use of CEST MRI. 24) A compound selected among:DOTAMidrazide, a chelate complex thereof with a paramagnetic metal ionselected from the group consisting of: iron (II) (high spin), iron(III), cobalt (II), copper (II), nickel (II), praseodymium(III),neodimium (III), dysprosium (III), erbium (III), terbium (III), holmium(III), thulium (III), ytterbium (III), and europium (III); the(bisDOTAMGLy) dimeric chelating ligand and the chelated complexesthereof with two transition or Ln(III) metal ions of the above group,and their physiologically acceptable salts.